The present invention relates generally to methods for the rapid quantitation of cells. More specifically, the present invention involves staining viable cells in a sample with a fluorescent dye and measuring the fluorescence.
The ability to quantify viable cells is vitally important to the food, pharmaceutical, environmental, manufacturing and clinical industries. Several methods are currently employed for the quantitation of viable cells. These methods include, but are not limited to, the standard plate count, dye reduction and exclusion methods, electrometric techniques, microscopy, flow cytometry, bioluminescence, and turbidity.
The standard plate count allows the enumeration of viable cells (or clumps of cells) also known as colony forming units (cfu) when the cells are grown on the appropriate medium under growth conditions. Atlas, R. M. and Bartha, R., Microbial Ecology, Addison Wesley Longman, New York, (1998). Current standards of live organism counts are based on the standard plate count, particularly in the food industry. However, colony counts are difficult to interpret since bacteria often clump or form chains that can give rise to significantly inaccurate estimations of the total number of live organisms in a sample. Also, bacteria, for example, can be in a xe2x80x9cmetabolically damagedxe2x80x9d state and not form countable colonies on a given medium. This is a greater problem when selective media are used. Thus, the standard plate count does not provide a definitive count of viable cells in a sample. Given these factors, such testing also requires skilled technicians who can distinguish separate cfus and who can aid in selecting appropriate growth medium. Moreover, the technique is not useful when rapid determination of cell counts is required since it often requires over 24 hours to obtain plate count results.
Dye reduction methods rely on the cell of interest to oxidize or reduce a particular dye. Harrington, W. F., (1998) Laboratory Methods in Food Microbiology, Academic Press, San Diego. These methods measure the activity of metabolically active organisms rather than provide an accurate measure of the total number of viable cells in a sample. Dyes, such as methylene blue, coupled with microscopic counting, are routinely employed to determine the relative number of microorganisms. This technique is widely employed but nevertheless suffers from factors that must be held constant during the assay, i.e., medium used, chemical conditions, temperature and the types of cells being examined. Also, dye reduction tests that incorporate microscopic counting techniques require trained technical personnel and often depend upon subjective interpretations.
Dye exclusion methods of cell quantitation depend on the ability of living cells to pump dye out of the cell and into the surrounding fluid medium. While dye may enter the interior of both live and dead cells, dead cells are not capable of actively pumping the dye out under the conditions and methods normally used. Dye exclusion is commonly used to enumerate animal, fungal and yeast cells. It is a method requiring skill, correct timing and correct choice of dye. It is not applicable to certain microbes and it yields incorrect live counts with stressed cells.
Electrometric methods such as impedance measurement indirectly determine the number of viable cells by measuring changes in the conductance of the growth medium following a period of cell growth. Stannard, C. J., Pettit, S. B., Skinner, F. A. (1989) Rapid Microbiological Methods for Foods, Beverages and Pharmaceuticals (ed. C. J. Stannard, S. B. Pettit and F. A. Skinner) Oxford: Blackwell Scientific. They are rapid tests that depend on variables that must be held constant and require expensive instrumentation. The Bactometer, by bioMerieux (France), is an example of an impedance instrument system designed for making estimates of viable cells in a sample.
Microscopic techniques typically involve counting a dilution of cells on a calibrated microscopic grid, such as a hemocytometer. A recent improvement in this technique is the direct epifluorescent filter technique (DEFT). Pettipher, G. L., Kroll, R. G. Farr, L. J. (1989) Rapid Microbiological Methods for Foods, Beverages and Pharmaceuticals (ed. C. J. Stannard, S. B. Pettit and F. A. Skinner) Oxford: Blackwell Scientific. In this technique, samples are filtered through a membrane filter that traps the cells to be counted. A fluorescent dye is attached to the cells and they are illuminated with ultraviolet light and counted. The technique requires an expensive microscope (an epifluorescent instrument) and a trained individual or an expensive automated system.
Flow cytometry involves the differential fluorescent staining of cells suspended in a relatively clear fluid stream of relatively low viscosity. The cell suspension is mixed with the fluorescent dye and illuminated in a flow cell by a laser or other light source. Labeled cells are detected automatically by a fluorescence detector focused on the cell. Brailsford, M. A. and Gatley, S. (1993) New Techniques in Food and Beverage Microbiology (ed. R. G. Kroll, A. Gilmour and M. Sussman Oxford: Blackwell Scientific. Pinder, A. C., Edwards, C. Clarke, R. G. (1993) New Techniques in Food and Beverage Microbiology (ed . R. G. Kroll. A. Gilmour and M. Sussman Oxford: Blackwell Scientific. The technique requires, and is limited by, expensive equipment. Some flow cytometric devices have been used by the food and dairy industry, but their application is limited by the high cost of instrumentation.
Bioluminescence has been routinely employed in the food sanitation industry to detect and quantify live organisms and cells. A common method employs the use of luciferin-luciferase to detect the presence of ATP. Harrington, W. F., (1998) Laboratory Methods in Food Microbiology, Academic Press, San Diego. Griffith. C. J., Blucher, A., Fleri, J. (1994) Food Science and Technology Today 8: 209-216. When used to quantitate cells, the technique depends on the assumption that there is a constant amount of ATP in a viable cell. ATP levels vary in a single cell over more than two orders of magnitude, making this method a relatively inaccurate technique for the enumeration of live organisms in a sample.
Turbidity of a liquid sample can be measured as an indication of the concentration of cells due to the light scattering/absorbing qualities of suspended cells. Harrington, W. F., (1998) Laboratory Methods in Food Microbiology, Academic Press, San Diego. The method is old but is still employed to estimate the bacterial concentration in a sample. The method is rapid and simple but is highly inaccurate since all cells, particles and substances, including non-living particulate matter, interfere with the interpretation of the results. Thus, while extremely beneficial under laboratory growth conditions where only one particular species is in the sample, it is usually not used for quantifying cells in a sample with multiple particulate species.
The present invention for the quantitation of cells is designed to overcome at least three problems that have been identified within the field. First, the technology circumvents the need for training personnel in how to plate, grow and count viable cells from colonies on agar plates. It also eliminates nearly all training and maintenance costs associated with most of the other methods. Second, the invention substantially decreases the time needed for cellular (bacteria) quantifications. Under current methodologies, quantification requires from 24 to 72 hours (plate counts and enrichment cultures), while the present invention permits more accurate quantitation in less than 20 minutes. Third, the technology offers substantial cost savings over existing methods of cellular quantitation.
Briefly, the present invention describes methods and kits for quantifying viable cells in a sample using fluorescent dyes that can be actively internalized by viable cells and have fluorescence properties measurably altered when bound to target components.
In one aspect of the present invention, a method for quantifying viable cells in a sample is disclosed, comprising the following steps: (1) contacting a sample with a fluorescent dye, wherein the dye is actively internalized by the cells and has fluorescence properties that are measurably altered when bound to target components; (2) detecting total fluorescence of the sample; and (3) correlating the total fluorescence to the number of cells in the sample. Within certain embodiments, (1) the cells in the sample are bacteria; (2) the fluorescent dye binds to DNA of the viable cells; (3) the sample is treated with DNase before it is mixed with the fluorescent dye; (4) the sample is treated with an agent that affects a cell membrane property of the cells (e.g., a detergent) prior to, subsequently or concurrently with the fluorescent dye; or (5) the fluorescent dye is PicoGreen(trademark).
In another aspect of the present invention, a method for measuring the number of live bacteria in a sample with a fluorescent dye is disclosed. The fluorescent dye used needs to be actively internalized by the bacteria and bind to DNA or other specific cellular components. When so bound, the fluorescence properties of the dye are measurably altered. The disclosed method comprises the following steps: (1) adding the fluorescent dye to a fraction of known volume of the sample; (2) measuring the total fluorescence and fluorescence properties of the fraction of the sample; and (3) correlating the total fluorescence to the number of bacteria in the fraction of the sample. Within certain embodiments, the fraction of the sample is treated with DNase or an agent that affects a cell membrane property of the cells (e.g., a detergent) before it is mixed with the fluorescent dye. Within other embodiments, PicoGreen(trademark) is used as the fluorescent dye.
The present invention also discloses kits for quantifying viable cells. One such kit comprises a cell suspension solution, a fluorescent dye that can be actively internalized by viable cells, and instruction for detecting dye binding to cellular components and correlating the binding to colony forming units. The cell suspension solution may include a DNase or an agent that affects cell membrane property, such as a detergent. In certain embodiments, the fluorescent dye may be PicoGreen(trademark) (Molecular Probes, OR). Another kit for quantifying viable cells in a sample comprises a first solution, means for mixing the first solution with the sample, means for concentrating the cells, a second solution containing a fluorescent dye that can be actively internalized by the viable cells and binds to DNA or other specific cellular components, means for mixing the second solution with concentrated cells, means for illuminating the resulting mixture with excitation light, measuring fluorescence emitted, and thereby determining the amount of DNA or other bacterial cellular components that the fluorescent dye binds to, and thereby providing a fluorescence value proportional to the number of bacteria in the sample. Upon binding, the fluorescence properties of the fluorescent dye are altered to a measurable degree. The first solution may contain a DNase or an agent that affects a bacterial cell membrane property, such as a detergent. Within certain embodiments, the fluorescent dye may be PicoGreen(trademark).
A method for measuring the ratio of viable cells to dead cells in a sample is also disclosed. The method may contain the following steps: saturating the sample with an internalizing first fluorescent dye, adding to the sample a second fluorescent dye that has an emission wavelength overlapped with that of the first fluorescent dye and is actively internalized by the viable cells, and detecting the fluorescence quenching of the first fluorescent dye. Either the first fluorescent dye or the second fluorescent dye may be PicoGreen(trademark) in certain embodiments.